Our primary objective is to sequence filtrate tetanus toxin. We will purify the toxin from culture filtrates and subsequent to breaking the disulfide bonds, fractionate the toxin into its heavy and light chain components. Each peptide will then be fragmented by cleavage with cyanogen bromide (8 peptides from the heavy chain and perhaps 5 peptides from the light chain). The peptides will also be fragmented by trypsin which should yield an appreciably larger number of peptides than cyanogen bromide. The peptides from each fragmentation procedure will then be separated and sequenced. If a peptide is too large for overlap other enzymes will be used. Toxin prepared from both cell extracts and culture filtrates will be prepared. The amino acid composition of peptides, prepared by cyanogen bromide and trypsin cleavage from each toxin preparation will be compared. This should detect any difference in the two toxins. There is no apparent reason to sequence toxin prepared by both methods. These experiments should allow us to compare the amino acid sequence of this unusual protein with its secondary structure and that of other nontoxic soluble proteins. It should also enhance our understanding of the molecular mechanism of the pathology of tetanus and ultimately aid in the prevention and care of this deadly disease.